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Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination

Published online by Cambridge University Press:  22 July 2015

Young-Hee Jeong ,
Yeong Ji Kim ,
Eun Young Kim ,
Se Eun Kim ,
Jiwoo Kim ,
Min Jee Park ,
Hong-Gu Lee ,
Se Pill Park  and
Man-Jong Kang
Young-Hee Jeong
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Yeong Ji Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Eun Young Kim
Affiliation:
Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Mirae Cell Bio Research Institute, Mirae Cell Bio Inc., Seoul, Korea.
Se Eun Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Jiwoo Kim
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
Min Jee Park
Affiliation:
Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Mirae Cell Bio Research Institute, Mirae Cell Bio Inc., Seoul, Korea.
Hong-Gu Lee
Affiliation:
Department of Animal Biotechnology, KonKuk University, Seoul, Korea.
Se Pill Park*
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea, Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea.
Man-Jong Kang*
Affiliation:
Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea, Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea
*
Se Pill Park. Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju, Korea. Tel: +82 64 754 4650. Fax: +82 2 6455 8759. E-mail: sppark@jejunu.ac.kr
All correspondence to: Man-Jong Kang. Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Korea. Tel: +82 62 530 2113. Fax: +82 62 530 2129. e-mail: mjkang@jnu.ac.kr
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Summary

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

Keywords

β-Casein geneCRISPR/Cas9 systemKnock-inTransgenic cattle
Type
Research Article
Information
Zygote , Volume 24 , Issue 3 , June 2016 , pp. 442 - 456
Copyright
Copyright © Cambridge University Press 2015 

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Footnotes

6

These authors contributed equally to this work.

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